PROJECT ABSTRACT/SUMMARY The goal of the proposed research is to determine the mechanisms by which the large, molecular weight immunophilin FKBP51 and the protein phosphatase 5 (PP5C) regulate the ISOC store-operated calcium entry channel in pulmonary endothelial cells. This project is of clinical significance because activation of ISOC is a key step leading to the formation of inter-endothelial cell gaps and increased permeability across the endothelial barrier. Increased permeability is a vascular event that occurs in both acute and chronic inflammation, yet effective therapies to prevent or reverse inter-endothelial cell gap formation are lacking. We have shown that the PP5C-FKBP51 axis inhibits ISOC and protects the endothelial barrier from calcium entry-induced disruption. However, the mechanism by which this axis inhibits ISOC is unknown. Thus, we seek to determine the mechanism by which the PP5C-FKBP51 axis inhibits ISOC. In this proposal we will determine whether PP5C and FKBP51 work together to inhibit ISOC through cytoskeletal rearrangement. Specifically we will test the hypothesis that PP5C and FKBP51 stabilize microtubules near the ISOC channel increasing a microtubule ? protein 4.1 interaction leading to disruption of the protein 4.1 ? spectrin interaction, which is critical for ISOC function. If found to be true, this will be a novel mechanism of ion channel regulation. Second, while we know that PP5C is required for the FKBP51-mediated inhibition of ISOC, we do not know how PP5C activity is regulated. S100 proteins are calcium-activated proteins that have been shown to activate PP5C, and are expressed in endothelial cells. We will test the hypothesis that calcium entry through the ISOC channel provides calcium needed for activation of S100A6 which will in turn activate PP5C allowing for inhibition of ISOC. If found to be true, this will represent a novel role of feedback inhibition by S100 proteins. Specifically, in vitro, in situ and in vivo experimental models will be used to determine: the mechanism by which PP5C and FKBP51 inhibit ISOC (Aim 1); whether calcium entry through ISOC activates S100A6 which in turn activates PP5C (Aim 2); and whether the-PP5C-FKBP51 axis is important for limiting inflammation-associated vascular permeability (Aim 3). Techniques to be used include: differential fractionation, immunoprecipitation, PLA and FRET to determine protein-protein interactions; fluorescence microscopy and patch-clamp electrophysiology to measure calcium channel function; video microscopy, biotin gap assay and resistance measurements to measure inter-endothelial cell gap formation in vitro; and the isolated, perfused lung model with Kf, extravascular lung water and albumin permeation measurements to determine permeability in situ. Finally, a systemic LPS model of acute lung injury will be employed to determine in vivo relevance.